The long non-coding RNA Dali is an epigenetic regulator of neural differentiation
- University of Oxford, United Kingdom
Figures
Figure 1 with 2 supplements
Conservation and expression within the Dali and Pou3f3 loci.
(A) Schematic illustration of the mouse Pou3f3 genomic region showing coding and non-coding transcripts, enhancer elements from Vista Enhancer Browser (Visel et al., 2007), CpG islands, and …
Figure 1—figure supplement 1
Analysis of the mouse and human Dali loci.
(A) A detailed view of the mouse Dali locus (red) indicating regions of vertebrate DNA sequence conservation. Full-length Dali transcript was mapped using Rapid Amplification of cDNA Ends (RACE) in …
Figure 1—figure supplement 2
The Pou3f3 locus occurs in a folded nuclear conformation both prior to and after the onset of the expression of its transcripts.
(A) Schematic representation of the Pou3f3 locus showing start sites of transcripts in the region and positions of recognition sites of the BglII restriction endonuclease (small vertical bars) used …
Figure 2 with 1 supplement
Dali plays a role in regulating genes in neuronal cells.
(A) qRT-PCR analysis validates reduced levels of Dali in three clonal Dali knockdown cell lines compared to a control line. Mean values ± s.e., n = 3. (B) Reduced neurite outgrowth in …
Figure 2—figure supplement 1
Non-coding transcripts in the Pou3f3 locus form a network of regulatory interactions.
(A) Dali regulates expression of lncRNA AK011913. N2A cells were transfected with either a non-targeting control (scrambled) or three independent Dali targeting shRNA expression vectors. Dali and …
Figure 3
Dali regulates transcription in both Pou3f3-dependent and -independent manners.
(A) N2A cells were transfected with either a non-targeting control (scrambled) or a Pou3f3 targeting shRNA expression vector (knockdown). Pou3f3 and Dali levels were measured by qRT-PCR 72 hr post- …
Figure 4 with 1 supplement
Gene expression analysis of stable Dali knockdown cells.
(A) Stable Dali knockdown resulted in statistically significant changes in the expression of 747 genes in N2A cells (1.3-fold, 5% FDR, Supplementary file 4). 332 genes were up-regulated, 415 …
Figure 4—figure supplement 1
Transcriptomics.
(A and C) Heatmap displaying expression changes in control (A) and stable Dali knockdown (C) cells treated with RA for 72 hr. (B and D) GO-analysis of genes differentially expressed (≥1.3-fold, 5% …
Figure 5 with 1 supplement
CHART-Seq analysis of Dali genomic binding sites.
(A) Peaks were called against control CHART-seq experiments and input DNA. We consider only the 1427 peaks common to both comparisons (Supplementary File 5). (B) Sequencing of Dali bound DNA reveals …
Figure 5—figure supplement 1
CHART Analysis.
(A) CHART-seq was performed using cocktails of capture (C-) oligos oligonucleotides complementary to accessible (violet) and/or evolutionary conserved (blue) regions of Dali and a non-targeting …
Figure 6
Dali associates with chromatin and transcriptional regulatory proteins.
Dali interacts with BRG1, SIN3A, and P66beta in mouse N2A cells (A) and DNMT1 in mouse N2A and human SH-SY5Y cells (B). Nuclear extracts prepared from UV cross-linked cells were immuno-precipitated …
Figure 7 with 1 supplement
Dali modulates DNA methylation at bound and regulated promoters.
(A) DNA methylation status of three CGI-associated promoters bound and regulated by Dali was assessed using bisulfite sequencing in control and two stable Dali knockdown lines. DNA methylation …
Figure 7—figure supplement 1
DNA Methylation analysis.
(A) DNA methylation status of three Dali-bound and regulated CGI-associated promoters (Dlgap5, Nos1, and Hmgb2; see Figure 7) was assayed in a stable control and two independently isolated Dali …
Additional files
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Supplementary file 1
Oligonuleotides.
- https://doi.org/10.7554/eLife.04530.016
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Supplementary file 2
Transient Dali knockdown microarray profiling.
- https://doi.org/10.7554/eLife.04530.017
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Supplementary file 3
Genes changing in transient Pou3f3 knockdown and intersection with Dali targets.
- https://doi.org/10.7554/eLife.04530.018
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Supplementary file 4
Stable Dali knockdown gene lists.
- https://doi.org/10.7554/eLife.04530.019
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Supplementary file 5
CHART analysis.
- https://doi.org/10.7554/eLife.04530.020
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Supplementary file 6
Motif discovery.
- https://doi.org/10.7554/eLife.04530.021
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Supplementary File 7
Mass spectrometry identification of Dali associated proteins.
- https://doi.org/10.7554/eLife.04530.022
